This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The fluid mosaic model played an important role for understanding the dynamic behavior in biomembranes. The model postulated that the membranous proteins are diffusing and/or fluctuating in a homogeneous lipid medium, i.e. two-dimensional liquid. However, recent studies on biomembranes have revealed that physiological function is firmly related to the heterogeneous environment in the lipid membranes. When some physico-chemical stimuli occur, such as interactions with ions, proteins, and sterols, then heterogeneous phases frequently appear in biomembranes, yielding microdomains, which have been termed lipid rafts. In fact, in model bilayer membranes of lipids and cholesterol (Chol), one can find liquid ordered (Lo) phases as well as liquid disordered or liquid crystalline (Lc) phases. We have developed 2D-ELDOR for measuring rotational diffusion as well as translational diffusion in lipid vesicle membranes. Our stud ies revealed the dynamic structures of the Lo and Lc phases in membranes of pure lipids (Lc) and lipid/Cholesterol (Lo), in which a chain labeled lipid a headgroup labeled lipid, or a spin labeled cholestane CSL was incorporated. Creation and annihilation of the spectral cross-peaks was observed in the spectra from Lc and from Lo phases respectively. Thus, visual inspection of cross peaks in the 2D-ELDOR spectra provides a convenient way to distinguish them. In the Lo phase, acyl chains undergo a faster rotational diffusion and higher ordering than in the Lc phase. On the other hand, the choline headgroup in the Lo phase exhibits faster motion and reduced ordering as compared to the Lc phase. Also, the translational diffusion rates in the Lo phase are significantly reduced in the presence of Chol. Thus 2D-ELDOR is demonstrated to be a powerful method to study the dynamic structure of membranes. This work has been published and extensive studies as a function of cholesterol concentration have been completed.